−E-2-(3,4-dichlorocinnamyl)-1-cyclopropylmethylpiperidine, and its antidiarrhoeal use

ABSTRACT

(−)E-2-(3,4-dichlorocinnamyl)-1-cyclo-propylmethyl-piperidine of formula I:                    
     and its pharmaceutically acceptable salts, is useful for treating diarrhea in mammals. Also disclosed are pharmaceutical compositions, processes for preparing compounds of formula (I) and intermediates useful for preparing compounds of formula I.

FIELD OF THE INVENTION

The invention relates to(−)E-2-(3,4-dichlorocinnamyl)-1-cyclopropylmethylpiperidine, its salts,and their use as antidiarrhoeal medicaments in man.

PRIOR ART

The secretory anomalies of the gastro-intestinal tract are responsible,with motor disorders, for the majority of chronic or acute diarrhoeaswhich, in 1990, were estimated to be the second cause of globalmortality, especially in child populations of developing countries.

Chronic diarrhoeas are defined by their generally persistent duration ofover two weeks. Their known mechanisms and the diagnostic strategy to beadopted in view of these cases has been documented by M. CERF—Gastroenterol. Clin. Biol, 1992, 16, T 12-T 21. and more recently by M.J. G. FARTHING—Eur. J. of Gastroenterol. & Hepatol. 1996, 8:157-167. Theacute diarrhoeas, a great majority of which are of infectious origin,have likewise been documented by M. CERF and M. HAGIAGE: Diarrhéesaiguës d'origine infectieuse (Acute diarrhoeas of infectiousorigin),—Editions Techniques—Encycl. Méd. Chir. (Paris-France),Gastro-entérologie, 9061 A¹⁰, 1992, 20pp.; and H. L. DuPONT—Reviewarticle: infectious diarrhoea—Aliment. Pharmacol. Ther. 1994; 8:pp.3-13. Among other causes, the important role of toxinogenesis duringbacterial infection is discussed, and, especially, the expression of thepathogenic capacity by the synthesis of thermolabile or thermostablecytotoxins and enterotoxins, which are responsible for secretorydiarrhoeas with a hydroelectrolytic component, and whose representativephysiopathological model is that of cholera. Other infectious agents areknown to cause diarrhoeas of this type, such as Salmonella, EscherichiaColi (E. coli) and Clostridium difficile (C. difficile) strains.

These latter agents, and more particularly C. difficile, are responsiblefor chronic and abundant secretory diarrhoeas, often of nosocomialorigin, in subjects submitted to an intensive antibiotic therapy such asHIV positive patients. In the latter, the particularly incapacitatingdiarrhoeas are often associated with malabsorption, and contribute tothe rapid development of an alarming state of denutrition.

For the treatment of secretory diarrhoeas, rehydration of the patientsis recommended and sometimes turns out to be essential. Some compoundshave been shown to be active (phenothiazine, chlonidine, bismuth salts)but their sensitive employment because of their secondary effects hasled to their generalization being abandoned. The usual symptomatictreatments call for adsorbent compounds (Fuller's earth), modulators ofthe intestinal flora and, very widely, compounds called retardants,which are morphinomimetic antidiarrhoeals: loperamide (INN) anddiphenoxylate (INN), known inhibitors of the motility of the GI tract:and, in fact, of controversial if not inadvisable utility for certainailments, among other reasons through the delay which they contribute tothe natural evacuation of pathogenic bacteria.

More recently, it has been proposed to treat these diarrhoeas withacetorphan (INN), a synthetic enkephalinase inhibitor dipeptide withantisecretory effect, which maintains the effect of enkephalins,antisecretory endogenous neuropeptides of the intestinal wall, which arenormally rapidly hydrolysed in vivo by the enkephalinases which makestheir effect fleeting.

As far as the therapy of diarrhoeas of patients infected with HIV isconcerned, it is frequently necessary to resort to serious methods,which can only be carried out in an inpatient environment, such asrehydration and renutrition by the enteral or parenteral routes, whichare combined with the symptomatic antidiarrhoeal treatment and anantibiotic therapy directed against the possible pathogenic agent. Theusual antidiarrhoeal agents only have, most often, a relative andepisodic efficacy. Recently, for these ciiarrhoeas and, more generally,cases resistant to conventional therapy, peptides inhibiting motilityand gastrointestinal secretion related to somatostatin have beenproposed (M. CAMILLERI—Digestion 1996;57 (suppl 1): 90-92 and M. J. G.FARTHING—Digestion 1996;57 (suppl 1): 107-113). Substitute syntheticcompounds for this endogenous mediator are octreotide (INN) andvaltreotide (INN), both octapeptides proposed with some success for thetreatment of secretory diarrhoeas of AIDS. Although their duration ofaction is considerably longer than that of somatostatin, these expensivecompounds are only active by repeated administration parenterally whichleads to prohibitive treatment costs and, because of their mode ofadministration, makes their use virtually impossible in an outpatientenvironment. In addition, their lack of specificity, which has beenpointed out, can involve secondary effects which dramatically aggravatethe state of denutrition of the patients (disorders of the regulation ofhydrocarbon metabolism and increase in steatorrhoea).

In addition, certain compounds defined as specific ligands for sigmareceptors have shown antisecretory properties suggesting their use inthe treatment of diarrhoeas. Thus(+)-N-cyclopropylmethyl-N-methyldiphenyl-1,4-ethyl-1-buten-3-yl-1-amine,or igmesine (INN), and its hydrochloride are disclosed among othercompounds in European Patent 0 362 001. The compounds of this patent aredefined in vitro as specific ligands for sigma receptors and shown, invivo, in rats, to be inhibitors of amnesic phenomena caused byscopolamine, and inhibitors of gastroduodenal ulcers caused by theadministration of cysteamine, this last activity being connected withtheir capacity to increase the alkaline duodenal secretion inanaesth,etized animals. In the broad sense, the compounds of this patentare indicated as useful for the treatment of dysfunctions of thegastrointestinal tract such as disorders of peristalsis, of motility,the phenomena of gastro-oesophageal and gastroduodenal reflux as well asfor gastric and gastroduodenal ulceration.

Subsequently to these studies, the sigma receptors, whose localizationwas known in the central nervous system and the immune system, have beendemonstrated by F. ROMAN et al. in the gastrointestinal tract of theguinea-pig (Life Sciences 1988, 42, 2217-2222) and then of man(Gastroenterology 1991, 100, A662).

In connection with these localizations, various experiments, amongothers by J. L. JUNIEN et al. (Neuropharmacology, Volume 30, No. 10,October 1991, pp. 1119-1124) have demonstrated the inhibitory action ofigmesine on colonic hypermotility induced by stress via thecorticotropin releasing hormone (CRH or CRF) in man. In addition,, P.RIVIERE et al. (Gastroenterol. Clin. Biol. 1991, 15 (2B), A70) show invitro that igmesine modifies the transmembrane ionic transport throughportions of mouse jejunum. This effect, antagonized by haloperidol,involves the participation of sigma receptors. V. J. CARLISI et al.(FASEB J. 1992, 6 (4), A1287) studied the effect of igmesine in vivo inmice, in a model of inflammatory diarrhoea caused by PGE₂: at a dose of30 mg/kg, igmesine, co-administered by the i.p. route with PGE₂, delayedby approximately 15 min the appearance of the diarrhoea, an inhibitoryeffect antagonized by haloperidol, and which, tested for by the oralroute, turned out to be zero at a dose of 60 mg/kg. More recently, G.SHI et al. (UEGW 1996—Paris—abstract No. 0786) showed the effect ofigmesine at a dose of 200 mg p.o. in man on intestinal hypersecretioninduced by PGE₂.

On the other hand, the application WO 95/15948 discloses derivatives of2-arylalkenylazacycloalkanes as ligands for sigma receptors, a processfor their preparation and their application in therapeutics. Thecompounds, their isomers and their addition salts are proposed for thepreparation of antipsychotic medicaments and are useful ingastroenterology. The experimental section describes in Example 2Eracemic E-2-(3,4-dichlorocinnamyl)-1-cyclopropylmethylpiperidine and itshydrochloride and, in addition, mentions without specific experimentalresults that the compounds of the application are active on secretorydiarrhoeas induced in mice by Salmonella lipopolysaccharicle (LPS),which suggests their use in the treatment of secretory diarrhoeas ofvarying aetiologies.

Overcoming the difficulties and uncertainties of the prior art; as setout, the present invention proposes for the purposes of symptomatologictreatment of secretory diarrhoeas the use, in appropriate medicamentousforms, of a novel optically active compound, which is a ligand for sigmareceptors, and whose spectrum of antidiarrhoeal properties isparticularly remarkable and distinguishes it formally from the priorart.

SUMMARY OF THE INVENTION

The subject of the invention by way of novel compound is optically pure(−)E-2-(3,4-dichlorocinnamyl)-1-cyclopropylmethylpiperidine (I) offormula

its addition salts with pharmaceutically acceptable acids and a processfor their preparation.

It likewise relates, by way of medicaments, to(−)E2-(3,4-dichlorocinnamyl)-l-cyclopropylmethyl-piperidine (I) and its,addition salts as well as their use in the preparation of pharmaceuticalcompositions intended for the treatment of diarrhoeas. It also comprisesthe medicamentous compositions comprising by way of active principle(−)E-2(3,4-dichlorocinnamyl)-1-cyclopropylmethylpiperidine (I) or itsaddition salts in therapeutically efficacious quantity.

DETAILED DESCRIPTION OF THE INVENTION

In first place, the invention is directed at optically pure(−)E-2-(3,4-dichlorocinnamyl)-1-cyclopropylmethylpiperidine (I) and itsaddition salts with pharmaceutically acceptable acids.

Optically pure is understood as meaning that the product is practicallyfree of its optical antipode and is at least of an optical purity of 95%and, preferably, equal to or greater than 98% in levorotatory eutomer,this being determined by appropriate analytical means.

Pharmaceutically acceptable addition salts are understood as meaningthose inorganic or organic salts, and their possible isomers, shown tobe non-toxic in the therapeutically customary doses of which, forexample, a list is presented in J. Pharm. Sci., 1977, Volume 66, pp.119. Non-limiting examples are acetic, benzenesulphonic,camphorsulphonic, citric, ethanesulphonic, hydrdbromic, lactic, maleic,malic, methanesulphonic, mucic, nitric, pamoic, phosphoric, salicylic,stearic, succinic, sulphuric or tartaric acid and hydrochloric acid,which is preferred. In another aspect, the invention relates to aprocess for the preparation of optically pure(−)E-2-(3,4-dichlorocinnamyl)-1-cyclopropylmethylpiperidine (I) whichconsists either in resolving the corresponding racemic compounddescribed in Example 2E of the application WO 95/15948, or in carryingout the chemical synthesis starting from (+)E-2-(3,4-dichlorocinnamyl)piperidine (III), itself obtained, accordingto the application WO 95/15948, by resolution of the correspondingracemic compound described in preparation 2E of the same patentapplication.

Resolution of the racemic compound corresponding to the product of theinvention consists in using an optically active acid to obtain, with theracemate, diastereoisomeric addition salts which are separated bycrystallization, and from which the two resolved enantiomers aregenerated by appropriate treatment. Acids currently used for thepreparation of such salts are, as non-limiting examples, theenanticimers of α-phenylglycine, α-phenylalanine, malic, mandelic andtartaric acids, of camphanic acid or alternatively ofα-methoxy-α-trifluoromethylacetic acid. An alternative method ofresolution is the direct resolution of the racemic compound byhigh-performance liquid chromatography on a column containing, forexample, as stationary phase a cellulose polymer grafted with carbamategroups such as the phase CHIRACEL OD (Daicel) and carrying out anelution with hexane containing a small quantity of triethylamine.

However, the preferred process is an adaptation of the methodologydescribed in the application WO 95/15948 and consists in carrying outthe resolution of (+/−) E-2-(3,4-dichlorocinnamyl)piperidine via theeutomeric diastereoisomeric salt with N-acetyl-L-phenylalanine, which,purified and treated, leads to (+) E-2-(3,4-dichlorocinnamyl) piperidine(III), and then in acylating (III) with cyclopropanecarboxylic acid toobtain (+) E-2-(3,4-dichlorocinnamyl)-1-cyclopropanecarbonylpiperidine(II), and then, in reducing (II) with a metallic or organometallichydride to obtain(−)E-2-(3,4-dichlorocinnamyl)-1-cyclo-propylmethylpiperidine (I) ofsuitable optical purity and, optionally, in making a pharmaceuticallyacceptable salt:

At the resolution stage, the recovery and the purification of the (−)antipode of the intermediate compound (III) allows, according to thesame process, the distomer of the compound of the invention to beprepared, namely(+)E-2-(3,4-dichlorocinnamyl)-1-cyclopropylmethylpiperidine synthesizedas comparison product.

The compound which is the subject of the invention and its salts haveremarkable pharmacological properties, indicative of their usefulness inthe form of medicaments for the treatment of secretory diarrhoeas inman. Although in vitro its affinity for sigma receptors is notstereospecific but of comparable intensity to that of its antipode,totally unexpectedly, the(−)E-2-(3,4-dichlorocinnamyl)-1-cyclopropylmethylpiperidine eutomer (I)shows in vivo, in various representative models of toxigenic secretorydiarrhoea, a very strong and stereoselective antidiarrhoeal activity.

Thus, the levorotatory eutomer appears from 4 to more than 50 times moreactive, by the oral route in mice, than its parent racemate in themodels of secretory diarrhoea caused by Salmonella lipopolysaccharide(LPS), the thermostable toxin of E. coli and the A and B toxins of C.difficile, without acting on the transit. Likewise, the eutomer turnsout to be close to 50 times more active than the racemate by the oralroute, in rats, on the inhibition of the intestinal secretion caused bycholera toxin.

In mice, by the oral route in the models of toxigenic secretorydiarrhoeas which have just been presented, when it is compared topublished compounds or those potentially capable of treating secretorydiarrhoeas, the eutomer which is the subject of the invention turns outto be:

from 66 to more than 4000 times more active than loperamide,

from 75 to more than 1400 times more active than igmesine,

and, on diarrhoea caused by LPS, more than 6000 times more active thanacetorphan.

Finally, compared to the enantiomers described in the application WO95/15948 and which turn out to be equally active in vivo in diarrhoeacaused by LPS, the study as a function of the dose shows, for thecomparison products (−) E-2-cinnamyl-1-cyclopropylmethylpyrrolidine(Example 1.3 of WO 95/15948) and (−)E-2-cinnamyl-l-cyclopropylmethyl-piperidine (Example 2A.3), aprogressive effect which, after a maximum, decreases significantly,whereas, differently, the eutomer shows a progressive effect followed bya stage in which this activity is maintained, which is demonstrative ofa safety of medical use and of a therapeutic aid, contrary to thecomparison products for which the zone of active concentrations, ortherapeutic window, is narrow and leads to a dosage which is difficultto handle and thus to a risk of inefficacy.

These studies, expanded on in the experimental section, demonstrate theparticularly interesting antidiarrhoeal activity of(−)E-2-(3,4-dichlorocinnamyl)-1-cyclopropyl-methylpiperidine (I) and ofits salts and their utility in the preparation of pharmaceutical formswhich can be administered to mammals including humans by routesappropriate to the pathology and condition of the subject. Thus, themedicaments can be administered by the parenteral, transdermal ortransmucosal route in conventionally known forms. However, thepharmaceutical forms adapted to outpatient treatments are preferred andespecially those intended for administration by the oral route.

The pharmaceutical compositions according to the invention which containa therapeutically efficacious quantity of compound (I) or of one of itssalts, are appropriate for the treatment of diarrhoeas which may becommonplace, such as those of infants or of travellers, and which may beacute and/or persistent and of varying aetiology in which the secretorycomponent can just as well result from a decrease in absorption as fromintestinal hypersecret ion.

Thus, the compositions of the compound (I) are indicated for thetreatment of diarrhoeas of inflammatory origin (Crohn's disease,post-radiotherapy enterites), of obstructions by lymphoid hyperplasia oralternatively of anti-cancer chemotherapy.

Likewise, these compositions are appropriate for the symptomatictreatment of hypersecretory diarrhoeas such as those followingneuroendocrine tumour conditions (Zollinger-Ellison syndrome, VIPoma,somatostatinoma, carcinoid syndrome), of viral, including HIV, orbacterial infections, or even of congenital dysfunctions or those causedby cathartic drugs, and during hypersecretions of intestinalinflammatory syndromes.

The good tolerance to the product shown in the preliminary testsjustifies, for treatments of two to three weeks, a daily dosage of 5 to50 mg and, in exceptional cases, for aggressive treatments of shortduration up to 100 mg. However, the majority of diarrhoeal conditionstreated are improved by daily dosages of 10 to 30 mg, the product beingadministered by the oral route, divided into two to four administrationsper 24 hours.

The product is administered in various pharmaceutical forms, containingper unit from 1.25 to 25 mg of the compound (I) or of one of its salts,especially of its hydrochloride; these forms can be, as non-limitingexamples, tablets, coated tablets, capsules, gelules, powders,solutions, suspensions or gels.

For the so-called solid forms, the compound (I) or its salt mayrepresent from 1 to 90% by weight of the finished form, thepharmaceutically acceptable excipients representing from 99 to 10%. Forliquid forms or those considered as such, the active principle canrepresent from 0.1 to 10% by weight of the finished form, the liquidphase representing from 99.9 to 90% by weight.

EXPERIMENTAL SECTION EXAMPLE 1

(−)E-2-(3,4-dichlorocinnamyl)-1-cyclopropylmethylpiperidine (I) and itshydrochloride Stage a): (+) E-2-(3,4-dichlorocinnamyl)piperidine (III)

The compound is prepared by resolution of (+/−)E-2-(3,4-dichlorocinnamyl)piperidine via the eutomeric diastereoisomericsalt obtained with N-acetyl-L-phenylalanine. In a 500 ml round-bottomedflask equipped with a stirrer and a condenser in the reflux position,23.00 g (85.1 mmol) of the racemic compound (preparation E2 of WO95/15948) and 8.82 g (42.6 mmol) of N-acetyl-L-phenylalanine areintroduced into 325 ml of acetone.

The mixture is heated to reflux with stirring, and a solution isobtained which is progressively cooled and then kept at 20° C. for 16hours with stirring. The eutomeric salt is filtered (dry weight: 15.50g−m.p.=134-141° C.), the filtrate is rendered alkaline and extractedwith dichloromethane, and the residual oil from concentration (13.60 g -50.2 mmol) is salified in the same manner as above in 200 ml of acetonewith 6.25 g (30.1 mmol) of Nacetyl-D-phenylalanine. The distomeric saltis removed and the filtrate is treated by rendering alkaline to obtain7.15 g (26.5 mmol) of residual product which is salified in 150 ml ofacetone with 3.57 g (17.2 mmol) of N-acetyl-L-phenylalanine. A secondsupply of eutomeric salt 6.00 g m.p. 150-152° C. is thus obtained afterfiltration.

The combined eutomeric salts (21.50 g) are recrystallized forpurification in 60 ml of boiling water; 4.30 g of product recrystallizeat 20° C. (m.p.=151-152° C.)

The insoluble matter is recrystallized in 60 ml of boiling water; 10.9 gof product (m.p.=153-154° C.) recrystallize at 20° C. The salt issuspended in water, rendered alkaline with a solution of sodiumhydroxide and extracted with dichloromethane. The solvent is removed bydistillation in vacuo and on a water bath. 6.10 g of product areobtained in the form of a pale yellow oil. Yield=53% [α]_(D) ²⁰=+9.600(c=0.5; dichloromethane)

TLC and NMR in accordance (identical to the racemic starting product)

Stage b):(+)E-2-(3,4-dichlorocinnamyl)-1-cyclopropane-carbonylpiperidine (II)

In a 250 ml reactor equipped with a stirrer and protected from moisture,6.00 g (22.2 mmol) of (+) E-2-(3,4-dichlorocinnamyl)piperidine (III)obtained in the previous stage are introduced under a nitrogenatmosphere into 100 ml of dichloromethane dried over molecular sieves.3.40 ml or 2.47 g (24.4 mmol) of triethylamine are added to the paleyellow solution and, after cooling to 10° C., 2.20 ml or 2.55 g (24.4mmol) of cyclopropanecarbonyl chloride are added in 10 min. The mixtureis kept with stirring for 30 minutes at 20-25° C. and then extractedsuccessively with 100 ml of 10% ammonia, 100 ml of 10% hydrochloricacid, 100 ml of saturated sodium bicarbonate solution and finally 100 mlof water. After drying over sodium sulphate, the dichloroimethane isremoved by distillation in vacuo and on a water bath. The residualproduct is obtained in the pure state as a yellow oil. Weight: 7.50g−yield=100%−[α]_(D) ²⁰=+22.600 (c=0.5; dichloromethane)

TLC(CH₂Cl₂/MeOH 10% ammoniacal 90/10 v/v): Rf=0.90-1.00

Stage c): (−)E-2-(3,4-dichlorocinnamyl)-1-cyclopropyl-methylpiperidine(I)

Under a nitrogen atmosphere, protected from moisture and withoutexceeding 0° C., on the one hand a suspension of 2.65 g (69.5 mmol) oflithium aluminum hydride (LAH) is prepared in 40 ml of dry THF, and onthe other hand a solution of 2.99 g (22.4 m mol) of aluminum chloride in40 ml of dry diethyl ether. After 30 min contact for each preparation,the LAH/THF suspension is introduced in 10 min ago at 0° C. into theethereal solution of AlCl₃, then a solution of 7.40 g (21.9 mmol) of theamide (II) obtained in the previous stage in 30 ml of dry THF isintroduced at this temperature and in 10 min. After 30 min at 0° C., themixture is brought to reflux for 10 min and then cooled rapidly to 0° C.4.6 ml of 15% (w/v) NaOH solution and then 4.6 ml of water are thenadded dropwise with caution. After 30 min contact, the mixture isfiltered on a buchner provided with a bed of infusorial earth. Thefiltrate is concentrated in vacuo and on a water bath to obtain(−)E-2-(3,4-dichlorocinnamyl)-1-cyclopropylmethylpiperidine(I) in theform of a yellow oil which is verified pure by TLC. Weight: 3.2 g−yield:84.6%−[α]_(D) ²⁰=67.20 (MeOH, c=8.0)

TLC(CH₂Cl₂/MeOH 10% ammoniacal 90/10 v/v): Rf=0.65-0.80. 1H-NMR (CDI₃-TMS): δ (ppm) 0.00-0.20 (m, 2H) ; 0.40-0.70 (m, 2H); 0.70-1.10 (m, 1H);1.10-1.80 (m, 6H); 2.10-2.80 (m, 6H); 2.90-3.20 (m, 1H); 6.20-6.40 (m,2H); 7.10-7.50 (m, 3H).

optical purity>98%, determined by HPLC column 4.6×150 mm Chiracel ODH 5μm (Daicel) , elution with hexane containing 0.1% of triethylamine,t°=30° C.

Hydrochloride: The base is dissolved in 60 ml of dichloromethane, 7.0 mlof 5.5 N hydrochloric ether are added, and then the solvents are removedby distillation. The solid residue is crystallized by dissolution in anisopropanol/diethyl ether mixture. The white insoluble matter isfiltered and dried in vacuo to constant weight. Weight: 5.40g−yield=68%.

m.p.=182-183° C.−[α]_(D) ²⁰=−19.200 (c=0.5; CH₂Cl₂)

Analysis (C₁₈ H₂₄ Cl₃ N): % C, H, Cl, N in accordance;

IR (KEir): 3400, 2900, 2500, 1450, 1130, 1020, 990, 820, 800 cm⁻¹

According to this process, starting from the distomeric salt removed inthe resolution in stage a), recovery and purification allow the (−)antipode corresponding to the intermediate compound (III) to berecovered, from which the distomer of the compound of the invention isprepared, namely (+)E2-(3,4-dichlorocinnamyl)-1-cyclopropylmethylpiperidine, synthesized ascomparison product.

EXAMPLE 2

Toxicity Study

In animals, the toxicity study was carried out in mice by the oralroute. No mortality was noted up to the strongest dose studied, namely300 mg/kg. No toxic symptoms were evident at doses lower than 100 mg/kg.Starting from this dose, symptoms were observed such as respiratorydepression, trembling, convulsions, a state of prostration or oflethargy which were reversible in a lapse of time of 24 hours aftertreatment.

EXAMPLE 3

In vitro Pharmacological Study: Affinity for Sigma Receptors

The study of the capacity of interaction with the sigma receptors of theeutomer which is the subject of the invention, of its antipode and ofits parent racemic compound, was carried out by the determination oftheir binding to a rat brain membrane preparation, previously loadedwith a labelled ligand specific for sigma receptors, in this; case(+)[3H]-SKF 10,047. The technique used (described by Largent, B. L. etal., J. Pharmacol. Exp. Ther., 1986, vol. 238, pp. 739-748) consists inincubating, in solutions of variable concentrations of the test product,the membrane preparation previously loaded with (+)[3H]-SKF 10,047.After filtration, the radioactivity of the solution, which isrepresentative of the displacement of the ligand by the test: product,is measured. Results are expressed as ICSO of the test product , whichis the concentration allowing the tritiated ligand to be displaced from50% of its binding sites in the membrane preparation

Results:: The values obtained are presented in Table 1:

TABLE 1 In vitro study - σ affinity σ affinity: Test compound IC₅₀ (nM)(−)E 2-(3,4-dichlorocinnamyl)-1- 17.9 cyclopropylmethylpiperidine(I)(eutomer) (+)E 2-(3,4-dichlorocinnamyl)-1- 21.8cyclopropylmethylpiperidine (antipode) (+/−)E2-(3,4-dichlorocinnamyl)-1- 44.1 cyclopropylmethylpiperidine (racemate)

EXAMPLE 4

In vivo Pharmacological Studies

4-1)—inhibition of experimental toxogenic diarrhoeas Methods

a) diarrhoea induced by Salmonella lipoiolysaccharide (LPS):

The test is carried out in mice according to a working procedure,instigated by M. J. CANCIO et al., Gastroenterology November 1992, 103(5), 1437-43, which in the rat induces, by an endotoxin, alterations inthe transport of water and of electrolytes at the colon level.

working procedure: dBA₂ male mice (Iffa-credo, les Oncins, France)weighing between 20 and 25 g are placed in individual cages. Afteradaptation of the animals to their environment, the test product isadministered by the oral route in solution or in aqueous suspension andthen after one hour (to of the test) an injection of lipopolysaccharides(LPS) of Salmonella enteriditis (Sigma ref. L6761) is carried out in thetail vein at a rate of 15 mg/kg. A preweighed filter paper is thenplaced under each cage and the weight of faeces eliminated by theanimals in two hours (t₁₂₀) is determined. The effect of the testproduct is determined and is expressed as a percentage of inhibition ofthe weight of faeces at the dose considered with respect to the weightof faeces of a batch of control animals only having received LPS underthe same conditions. These results allow the ED₅₀ of the compounds to becalculated, which is the effective dose allowing 50% of the weight ofthe faeces caused by the administration of the diarrhoeal agent to beinhibited.

b)—diarrhoea induced by the thermostable toxin of E. coli: Nourishedmale NMRI mice (30-35 g) are weighed and placed in individual cagespreviously covered with white paper allowing the viewing of the faecalmatter voided. The faeces are recovered as soon as voided and arebrought together, by periods of 30 min, for 120 min. The faeces thusbrought together are weighed before (fresh weight) and after (dryweight) drying at 120° C. for 24 h. The quantity of water present in thefaeces is calculated as the difference (fresh weight—dry weight). Thethermostable toxin of Escherichia coli (Sigma, E5763) is administered bythe oral route at time zero at a dose of 600 U/mouse. The animals of thecontrol batch receive, at time zero, an oral administration of saline.The administration of the test products is carried out by the oral route1 h before the administration of the toxins. The results are expressedas the quantity of faecal water amassed at time 120 min, and allow theED₅₀ of the compounds to be calculated, which is the effective doseallowing 50% of weight of water of the faeces to be inhibited under theaction of the test product.

c)—diarrhoea induced by the A and B toxins of C. difficile: theexperimental protocol, the calculation and the expression of the resultsare identical to those described above. The A and B toxins of C.difficile are administered by the oral route at time zero at a dose of 6ng/mouse.

Results

i)—Compared inhibitory activity of the levorotatory eutomer (I) with the(+/−) parent racemate in mice, p.o administration

TABLE 2 Inhibitory activity ED₅₀ (mg/kg) Toxigenic Laevorotatory (+/−)parent diarrhoea eutomer (I) racemate Salmonella LPS 0.0056* 0.038 E.coil 0.019 0.077 C. difficile <0.001 0.058 *inhibitory effect notreversed by naloxone

ii)—compared inhibitory activity of the levorotatory eutomer (I) withloperamide (INN), igmesine (INN) and acetorphan (INN) in mice, p.oadministration

TABLE 3 Inhibitory activity: ED₅₀ (mg/kg) Toxigenic eutomer diarrhoea(I) loperamide igmesine acetorphan Salmonella LPS 0.0056 0.37 0.42 34 E.coli 0.019 3.92 1.72 N.T. C. difficile <0.001 4.27 1.45 N.T. N.T.: nottested

iii)—diarrhoea induced by Salmonella LPS: compared inhibitory activityof the levorotatory eutomer (I) with(−)E-2-cinnamyl-1-cyclopropylmethylpyrrolidine (Ex. 1.3 of WO 95/15948)and (−)E-2-cinnamyl-1-cyclopropylmethyl-piperidine (Example 2A.3) inmice, p.o. administration.

TABLE 4 Inhibition of diarrhoea (%) Dose Test product administeredLevorotatory (mg/kg) eutomer (I) Ex. 1.3 Ex. 2A.3 0.00001  29 0.00003 45 0.0001 65.8 0.001  7.1  27.9  36.8 0.003  46.1  63.8 0.01 59.7 78.8 29.5 0.03 59.5  46.7 0.1 69.5  12.7 ED₅₀ 0.0056 0.002 0.00004 (mg/kg)

These results demonstrate the therapeutic advantage provided by thecompound of the invention, as it shows a significant effect at a dose of0.003 mg/kg (46.1%), an effect which continues up to a dose of 0.1 mg/kgand beyond. Differently, the comparison compounds only manifest aworthwhile activity within narrow dose limits:

from 0.003 to 0.03 mg/kg (63.8 to 46.7%) for the compound of Example1.3,

from 0.00003 to 0.001 mg/kg (45 to 36.8%) for the compound of Example2A.3, the activity decreasing considerably at the higher doses for thesetwo compounds.

4-2)—inhibition of intestinal secretion induced by cholera toxin:so-called enteropoolinq technique.

Female Wistar rats (160-180 g) are made to fast from solid food 24 hbefore the test. At time zero, the animals receive, by the oral route,0.1 mg/kg of cholera toxin (Sigma, C3012). Three hours afterwards, theanimals are sacrificed by cervical dislocation. After median laparotomy,the intestine is ligated at the level of the pylorus and the ileocaecaljunction. It is then removed (from the duodenum to the caecum), andweighed full and them empty. The administration of the test products iscarried out by the oral route one hour before the administration of thecholera toxin. The results are expressed by weight of intestinalcontents and allow the ED₅₀ of the compounds to be calculated, which isthe effective dose allowing the weight of the intestinal contents to beinhibited by 50% under the action of the test product.

Results

TABLE 5 Inhibition of intestinal secretion induced by cholera toxin:ED₅₀ (mg/kg) Laboratory eutomer (I) (+/−) parent racemate 0.038 1.9

EXAMPLE 5

Pharmaceutical Forms

By way of illustration, the formulation and the preparation of thehydrochloride of the levorotatory eutomer (I) of the invention in theform of capsules and of tablets containing 40 mg of active principle perunit is presented.

5- 1 - Unit formulation of capsules and preparation:

Hydrochloride of the eutomer (I) 40.0 mg Lactose 59.3 mg Colloidalsilica 0.2 mg Magnesium stearate 0.5 mg Total 100 mg

The powders are intimately mixed and then distributed at a rate of 100mg per unit into capsules of appropriate size.

5 - 2 - Unit formulation of tablets and preparation:

Hydrochloride of the eutomer (I) 40.0 mg Lactose 72.0 mgHydroxypropylmethylcellulose (HPMC) 3.6 mg Crosslinkedcarboxymethylcellulose 3.6 mg (CMC) Colloidal silica 0.24 mg Magnesiumstearate 0.6 mg Total 120 mg

The active principle is mixed into the lactose and then granulated withthe HPMC in solution. The grains are dried and screened on a grid of 1mm mesh. The CMC and the silica are mixed and then added to thegranules. The mixture is then mixed intimately with the magnesiumstearate and then compressed at a rate of 120 mg per scored tablet.

What is claimed is:
 1. A compound according to formula I which is(−)E-2-(3,4-dichlorocinnamyl)-1-cyclopropylmethylpiperidine and itspharmaceutically acceptable acids.


2. The hydrochloride salt of claim
 1. 3. A process for preparing thecompound of claim 1 consisting of: (a) carrying out the resolution of(+/−) E-2-(3,4-dichlorocinnamyl)piperidine with N-acetyl-L-phenylalanineto provide (+) E-2-(3,4-dichlorocinnamyl)piperidine (III); (b) acylatingcompound (III) with cyclopropanecarbonyl chloride to obtain (+)E-2-(3,4-dichlorocinnamyl)-1-cyclopropanecarbonylpiperidine (II); (c)reducing compound (II) with a metallic or organometallic hydride toobtain (−) E-2-(3,4-dichlorocinnamyl)-1-cyclopropylmethylpiperidine (I);and optionally (d) making the salt of compound (I) with apharmaceutically acceptable acid.
 4. A method of treating diarrhea inmammal, comprising administering to a mammal in need thereof apharmaceutically acceptable amount of the compound of claim
 1. 5. Apharmaceutical composition for treating diarrhea in a mammal, comprisingthe compound of claim 1 admixed with a pharmaceutically acceptablecarrier or excipient.
 6. The compounds (+)E-2-(3,4-dichlorocinnamyl)piperidine (II) and (+)E-2-(3,4-dichlorocinnamyl)-1-cyclopropanecarbonylpiperidine (II) for usein preparing the compound of claim 1.